Abstract

Centromere associated protein-E (CENP-E), a mitotic checkpoint protein that is cell cycle-regulated and highly expressed in mitosis, is required for efficient, stable microtubule capture at kinetochores during mitosis. Absence of CENP-E results in misaligned chromosomes leading to metaphase arrest. In our studies, microtubule-stabilizing agents such as Taxol and epothilone B (EpoB) transiently mediate overexpression and phosphorylation of CENP-E in a variety of cancer cell lines. The association of CENP-E with microtubules is markedly reduced in drug-sensitive cells treated with Taxol or EpoB, compared to untreated cells, suggesting that phosphorylation of CENP-E inhibits its binding to microtubules. The CENP-E level in an EpoB-resistant A549 cell line, EpoB40, is ~ 2-fold higher than in A549 cells. To investigate the effect of CENP-E overexpression on drug sensitivity, a full-length CENP-E cDNA was transfected into HeLa cells. CENP-E overexpression did not alter Taxol or EpoB sensitivity. In contrast, suppression of CENP-E expression by CENP-E siRNA transfection resulted in an approximately 2-fold increase in drug sensitivity in drug-sensitive and -resistant cells, suggesting that a minimal quantity of CENP-E is required for maintaining its function. The mitotic spindles in EpoB40 cells were examined and it was found that the distance between the two centrosomes during metaphase was approximately 25% shorter than in A549 cells. The shorter spindles in EpoB40 cells indicate defects in the spindle-assembly checkpoint system in these cells. It is known that CENP-E binds to the checkpoint protein BubR1 and enhances recruitment of BubR1 to each unattached kinetochore and stimulates BubR1 kinase activity. Suppression of CENP-E caused a decrease in BubR1 levels in EpoB40 cells. Immunofluorescence studies indicated that targeting of CENP-E and BubR1 to the kinetochores during metaphase was significantly reduced in EpoB40 cells, compared to A549 cells, and colocalization of CENP-E and BubR1 at the kinetochores was not observed in EpoB40 cells. In addition, immunoprecipitation studies demonstrated that the interaction between CENP-E and BubR1 was diminished in EpoB40 cells and in a Taxol-resistant A549 cell line, AT-12. These observations suggest that defects in the mitotic checkpoint, including failure of CENP-E and BubR1 targeting to the kinetochores and a diminished interaction between these two proteins, have a role in the development of EpoB-mediated drug-resistance.